Lassa, Junin, Machupo, Guanarito, and Sabia viruses (family Arenaviridae) cause severe disease, particularly hemorrhagic fever, in humans. These 5 arenaviruses have been classified as Category A agents because they are considered to pose a significant risk to the security of the United States. Rapid, accurate diagnosis of acute disease caused by these viruses is critical to a timely and appropriate response by public health authorities in the event of an intentional exposure of civilians to infectious virus, naturally occurring human disease, and accidental exposure of laboratorians to infectious virus. At present, our ability to accurately diagnose arenaviral infections in a timely manner is limited and largely unproven. The broad objective of the proposed work is to assess the utility of various nucleic acid-based methodologies for rapid, accurate detection of arenaviral RNA. There are 3 specific aims. Aim #1 Generate a database of nucleocapsid protein (NP) and glycoprotein precursor (GPC) gene sequences representative of the genetic diversity of the Category A arenaviruses. Knowledge of the genetic diversity of these viruses is essential to rational design of oligonucleotide primers and probes for nucleic acid-based assays for arenaviral RNA. Aim #2 Develop conventional and fluorogenic probe-based RT-PCR assays for detection of LAS, JON, MAC, GTO, and SAB viral NP or GPC gene-specific RNA. Aim #3 Develop a DNA microarray for detection of cDNA generated from LAS, JUN, MAC, GTO, and SAB viral NF gene-specific RNA, and characterization of arenaviruses to the level of stereotype or species. The sensitivities of the RT-PCR assays and cDNA microarray will be measured against the success of virus isolation in vitro, as measured by plaque assay. The accuracy of each assay will be assessed further by testing strains of Lassa, Junin, Machupo, and Guanarito viruses, and strains of other arenaviruses (e.g., Amapari, Flexal) that were not used to develop or test the first generation assay.